Abstract 6277: Identification of ATR inhibitors as therapeutic opportunities in Desmoplastic small round cell tumors
Authors: Asuka KAWAI-KAWACHI, Madison LENORMAND, Clémence HENON, Thomas EYCHENNE, Leo COLMET-DAAGE, Nicolas DORVAULT, Marlene GARRIDO, Clémence ASTIER, Carine NGO, Helen PEMBERTON, Aditi GULATI, Stephen PETTITT, Roman CHABANON, Christopher J LORD, Sophie POSTEL-VINAY
Journal: Cancer Research (2022)

Abstract Introduction: Desmoplastic small round cell tumor (DSRCT) is a rare and very aggressive subtype of sarcoma, affecting predominantly young males. DSRCT is molecularly characterized by the t(11;22)(q24;q12) chromosomal translocation, which fuses EWSR1 to WT1 and encodes an aberrant chimeric transcription factor. A few additional secondary mutations have been reported, notably in genes encoding proteins involved in chromatin remodeling and DNA repair. No therapeutic advance have been done in the past 20 years, and the five-year overall survival is still below 15% in metastatic cases, despite aggressive poly-chemotherapy and extensive surgical debulking. Thus, we aimed to identify actionable targeted dependencies to propose novel therapeutic approaches, using drug screening and molecular biology. Material and Methods: Drug sensitivity screen evaluating 80 cancer-relevant small molecule inhibitors in dose-response was performed on the JN-DSRCT-1 (JN1) cell line. Revalidation was performed on JN1 and in a Champions Oncology PDX-derived in-house-generated DSRCT cell line (R) using multiple clinical compounds. Mechanistic dissection was performed using flow cytometry, immunofluorescence, western blotting, DNA fiber assay, RT-qPCR, and dot blot to evaluate markers of DNA damage response, replication stress and cell-autonomous innate immune signaling. Results: The drug screen identified that JN1 cells were particularly sensitive to one ATR inhibitor (ATRi) and multiple PARPi inhibitors (PARPi), which scored #6, 7, 9 and 20 of the hits. Sensitivity to ATRi was revalidated in the JN1 and R cell lines, whereas sensitivity to PARPi was only revalidated in the JN1 cell line - consistent with the very low PARP1 expression in that cell line. Sensitivity to ATRi and PARPi was partially reversed upon EWS-WT1 silencing. Combination experiments revealed that both drugs were synergistic in the JN1 cell line. Exposure to PARP inhibitor increased the number of γH2AX and RAD51 foci, suggesting that JN1 cells were not deficient in homologous recombination. Further, PARPi or ATRi exposure decreased replication fork speed and increased R-loops accumulation, whereas EWS-WT1 silencing increased replication fork speed. Resistance to PARPi and to ATRi was restored upon exogenous expression of the R-loop resolution enzyme RNAseH1. The combination of PARPi and ATRi also induced micronuclei formation and activated the cGAS-STING cytosolic innate immune sensing pathway, together with increased expression of CCL5, CXCL10 and PD-L1. Conclusion: DSRCT cells are sensitive to ATR and PARP inhibitors, through a mechanism that, at least in part, may involve enhanced replication stress and increased R-loop formation. Our data provide the preclinical rationale for assessing ATRi combined with PARPi in patients with DSRCT, as a cytotoxic therapy or as an immunomodulatory approach to enhance cell-autonomous immunogenicity. Citation Format: Asuka KAWAI-KAWACHI, Madison LENORMAND, Clémence HENON, Thomas EYCHENNE, Leo COLMET-DAAGE, Nicolas DORVAULT, Marlene GARRIDO, Clémence ASTIER, Carine NGO, Helen PEMBERTON, Aditi GULATI, Stephen PETTITT, Roman CHABANON, Christopher J LORD, Sophie POSTEL-VINAY. Identification of ATR inhibitors as therapeutic opportunities in Desmoplastic small round cell tumors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6277.